This invention relates to immunological and specific binding detection of biological particles; more particularly this invention relates to detection of biological particles such as antibody, hormone, specific binding protein, receptor and antigen by fluorescent and immunofluorescent dyes.
This invention relates to the subject matter of U.S. Pat. Nos. 4,011,308 "Method for Surface Immunological Detection of Biological Particles by the use of Tagged Antibodies," and 3,926,564 "Substrate for Immunological Tests and Method Fabrication Thereof," both granted to I. Giaever. Other publications related to the present invention are "Blood Coagulation Studies with the Recording Ellipsometer" by L. Vroman (National Bureau of Standards Miscellaneous Publication 256 September 1964); "A Study of Antigens and Antibodies by the Monolayer Film Technique of Langmuir" M. F. Shaffer and J. H. Dingle (Proceeding of Society of Experimental Biological Medicine 38, pages 528-530, 1938). "Immunological Reactions Between Film of Antigen and Antibody Molecule" by A. Rothen [Journal of Biological Chemistry Vol. 168 page 75-97 (April, May 1949)]; "The Beginnings of Immunofluorescence" by A. H. Coons (J. Immunology 87 pages 499-503 (1961)) "Fluorescent Protein Conjugates" by R. F. Steiner and H. Edelhock (Chemistry Review 62, pages 457-483 (1962), and "Radio-immunoassay" by D. S. Skeller et al. [Clinical Chemistry 19(2) pages 146-186 (1973)].
Immunological and specific binding reactions are highly specific biochemical reactions. The immunological reaction is vital in combatting diseases. The specific binding proteins and receptors are important in the transportation and balance of specific hormones, and molecules which affect the hormone function. To perform this kind of specific binding reaction on a metal surface, Shaffer et al., Rothen, and many other investigators have used ellipsometers to detect the amount of antibody bound to antigen or vice versa. Recently, Giaever has invented a visual detecting device using a specially prepared metal surface (see U.S. Pat. No. 3,926,564). Because the signal is detected by the naked eye, the quantitative determination is somewhat arbitrary.
According to this invention, a metal surface is used. No special preparation of the metal surface is needed. Most metals prepared by evaporation using a commercially available heating or sputtering device will meet the necessary requirement for this invention and produce a highly reflective metal surface. The metal is used to bind a monomolecular layer of protein, for example protein containing an antigen, while the amount of antibody (second biological particle) bound to this antigen is determined by the induced fluorescent emission from the fluorescent dye that is bound to an antibody (third biological particle) of this antibody.
The principal object of my invention is to provide an easy method and an apparatus to detect immunological or specific binding reactions. Another object of my invention is to provide an apparatus and an easy method to detect biological particles in a solution that may be serum, body secretion, body fluid, urine, tissue extraction etc. The biological particle may be a small particle like hormones, antibodies, plasma proteins, or a large particle like a virus, bacteria, cells, that are capable of stimulating antibody production. A further object of my invention is to provide an apparatus and simple method to perform diagnostic tests. In order to perform such a test, two appropriate biological particles with high mutual binding affinity must be found, one of them must be protein or protein bound (for example steroid hormone or polypeptide bound to bovine serum albumin) and the other a third biological particle, usually the antibody of the second biological particle. Because the novel apparatus according to the invention is used for quantitative signal detection, the metal surface need not be prepared from a particular kind of alloy or have a controlled thickness and the first biological particle, second biological particle and third biological need not form a layer so as to be detected. The preselected protein layer is absorbed on the surface of the substrate in a monomolecular layer (which includes first biological particle). When a suspect solution is tested for the presence or absence of the biological particle of interest (second biological particle), the monomolecular protein layer is placed in contact with the suspect solution for a sufficient long period of time to permit a specific binding reaction to occur. If the biological particle of interest is present, a specific reaction occurs between the initial protein layer and the biological particle of interest, resulting in some binding between them. This invention uses fluorescent or immunofluorescent stain to detect the amount of the second biological particle that is bound to the substrate. Immunofluorescent stain has long been used in histochemistry to detect the presence of antigen. In a traditional procedure, the antibody of the specific antigen is prepared and coupled with fluorescent dye. This fluorescent antibody is used as dye to stain a tissue slice, and a fluorescence microscope is used to visualize the existence of the specific antigen in the tissue. In this invention, we use the fluorescent antibody or specific binding protein to recognize the second biological particle that is bound to the first biological particle which is bound to the metal surface.